BRPI0707642A2 - Urine Gene Expression Rates for Cancer Detection - Google Patents

Abstract

EXPRESSION RATES OF URINE GENE FOR CANCER DETECTION. The present invention relates to methods for determining the presence of cancer in an individual based on analyzing the levels of expression of an underexpressed tumor marker (TM) and at least one other TM. Specifically, this invention relates to the determination of a cancer, particularly bladder cancer, by performing rate analysis, regression or classification of the levels of expression of at least one underexpressed TM, particularly an underexpressed bladder TM, and at least one overexpressed TM, particularly an overexpressed TM. In various aspects, the invention relates to kits and devices for carrying out these methods.

Description

Report of the Invention Patent for "URINE GENE DETECTION RATES FOR CANCER DETECTION".

TECHNICAL FIELD

This invention relates to cancer detection. Specifically, the invention relates to the use of markers for the detection of bladder cancer. More specifically, this invention relates to the use of an underexpressed pain marker in combination with at least one other marker for bladder cancer detection.

BACKGROUND

Cancer patient survival is greatly enhanced when cancer is treated early. For bladder cancer, patients diagnosed with early-stage disease have 5-year survival rates of> 90%, compared to approximately 15-30% for patients diagnosed with advanced disease. Therefore, developments that result in early diagnosis of bladder cancer may result in an improved prognosis for patients. The method established to detect bladder cancer using urine samples is cytology. However, cytology is known to be only about 75% sensitive to detect invasive bladder cancer and only about 25% sensitive to detect superficial bladder cancer (Lotan and Roehrborn, Urology 61, 109-118 (2003)).

Identifying urine cancer-specific markers can provide a valuable method for early cancer diagnosis, resulting in early treatment and improved prognosis. Specific cancer markers also provide a way to monitor disease progression, allowing the effectiveness of surgical, equi-radiotherapy treatments to be monitored.

At present, the safest method to detect bladder cancer is cystoscopy accompanied by histology of biopsied lesions. However, this technique is time consuming, invasive and its sensitivity is only about 90%, meaning that about 10 percent of cancers are not detected using these methods. Of noninvasive methodologies, urine cytology, which detects microscopically exfoliated malignant cells, is the preferred method today. Although cytology has a specificity of about 95%, it has poor sensitivity (9-25%) of low grade paralysis, is extremely dependent on sample quality, and suffers from high inter-observer variability.

Several urine protein markers are known. Tests for these markers offer better sensitivity than cytology, but tend to suffer from suboptimal specificity because elevated levels of these markers are also commonly observed in patients with nonmalignant diseases including inflammation, urolithiasis, and benign prostatic hyperplasia. For example, NMP22, which detects a specific nu-clear matrix protein, has a sensitivity of 47-87% and a specificity of 58-91%.

A disadvantage associated with urine testing is that individual marker levels may vary significantly with: (i) different urine collection methods (catheterized, vacuum, urine pellets); (ii) daytime urine sampling calculation; (iii) the sampling point during emptying (eg flow vs. final sample); and (iv) deurine concentration associated with variant fluid intake, renal function or diseases that affect plasma volume. These variations have the potential to result in false positive and false negative tests. Although some of these variations may be reduced using strict standard operating procedures, patient compliance with these procedures may be unreliable. The effect of variant urine concentration may in some cases be calculated by assessing marker levels for urinary creatinine, however, this increases the cost and complexity of the test, particularly when sample preparation or storage methods differ. marker and creatinine measurement.

There is a need for simple tools for early cancer detection and diagnosis. This invention also provides marker-based methods, devices, and kits, specifically bladder cancer marker rates, regression, or classification analysis, to aid in the detection and diagnosis of bladder cancer.

SUMMARY OF THE INVENTION

The present invention provides a method for determining the presence of a cancer in an individual, comprising:

(a) provide a sample of the individual;

(b) detecting the level of expression of at least two members of the tumor marker (TM) family in said sample, wherein at least one TM is an underexpressed TM;

(c) establish whether the patient has cancer according to a predetermined threshold.

Step (c) can be performed by determining the expression rate of said TMs, or by performing regression analysis or sorting on the expression levels of TM.

The TM can be a TM. The cancer to be detected may be bladder cancer, and in certain embodiments at least one of the TMs is an overexpressed TM. The overexpressed TM can be selected from the group outlined in figure 11 or figure 12.

In certain embodiments at least one subexpressed TM is a TM selected from the group outlined in Figure 3 or Figure 4.

In other embodiments of the present invention the detection step is performed by detecting TM mRNA overexpression, a TM protein, or a TM peptide.

The sample may be any of biopsy, blood, serum, peritoneal lavage, cerebrospinal fluid, urine and evacuation samples.

The present invention also provides a device for detecting a TM comprising:

a substrate having a TM capture reagent therein; and a detector associated with said substrate, said detector is capable of detecting a TM associated with said capture reagent, wherein the TM is an underexpressed TM.

The TM can be a TM.

The TM capture reagent may be an oligonucleotide or an antibody.

In certain embodiments, the TM may be a TM selected from the group outlined in Figure 3 or Figure 4.

The present invention also provides a kit for determining the presence of a cancer in an individual, comprising: a substrate;

at least two TM capture reagents, wherein at least one TM is an underexpressed TM; instructions for use.

The TM can be a TM.

The TM capture reagent may be a TM specific oligonucleotide or a TM specific antibody.

The TM detected by the kit can be a TM selected from the group outlined in figure 3 or figure 4.

At least one of the TMs detected by the kit may be an overexpressed TM or an overexpressed TM. The overexpressed TM can be selected from the group outlined in figure 11 or figure 12.

BRIEF DESCRIPTION OF THE FIGURES

This invention is described with reference to the specific embodiments thereof and with reference to the figures in which:

Figure 1 represents a table showing the urine sample characteristics used in qPCR analyzes.

Figure 2 is a table of oligo-nucleotide marker primers and probes for bladder cancer qPCR analysis according to the present invention.

Figure 3 is a table of subexpressive bladder tumor tumor markers identified using bladder cancer microarray methods.

Figure 4 is a table of subexpressive bladder tumor markers identified using microarray methods and bladder cancer samples that have negligible expression in whole blood but high expression in normal bladder tissue. fibrillation monocrystals showing the rates of three markers of transient depletion cell carcinoma (TCC) (HoxA13, IGFBP5, and MDK) with the subexpressive marker LTB4DH for urine samples from patients with nonmalignant urological disease or CBT. Boxes define the 25Q, 509 and 759 percentiles and horizontal bars express adjacent values. Discrepant values are shown by circles. Empty boxes represent samples of nonmalignant disease controls and shaded boxes represent samples of patients with CBT.

Figure 6 shows examples of TCC detection sensitivities and specificities by tests including LTB4DH. (a) single tests; (b) .tests in combination using LTB4DH and two of the threeHoxAI3, IGFBP5, and MDK markers.

Figure 7a-c shows ROC curves for the sensitivity and specificity of detecting CBT in urine samples using rates including LTB4DH. 7a. IGFBP5 / LTB4DH; 7b. MDK / LT4BDH; 7c. Ho-XA13 / LTB4DH.

Figure 8a-f shows dispersion plots for combination assays, a-c using LTB4DH and two of the three HoxAI3 markers, IGFBP5, and MDK, and d-f were repeated using BAG1 for LTB4DH. 8a.MDK / LTB4DH and IGFBP5 / LTB4DH; 8b. MDK / LTB4DH and HoxAI3 / LTB4DH IGFBP5 / LTB4DH and HoxAI3 / LTB4DH; 8d MDK / BAG1 and IGFBP5 / BAG1; 8e.MDK / BAG1 and HoxAI 3 / BAG1; 8f. IGFBP5 / BAG1 and HoxAI 3 / BAG1.

Figure 9a-b shows scatter plots showing the correlation between ACt ratios for IGFBP5 and ACt ratios for IGFBP5 / LTB4DH and urine creatinine concentration. 9a. urine samples from patients with CBT 9b. Urine samples from patients with nonmalignant disease

Figure 10a-f represents auto-auto dispersion plots showing the distribution of empty and catheterized urine samples from TCC patients analyzed using the bladder tumor marker MDK, IGFBP5 and HoxAI3 alone or in relationships with LTB4DH.

Figure 11 shows known overexpressed markers of invasive bladder tumors.

Figure 12 shows known overexpressed markers of superficial bladder tumors.

Figure 13 shows the clinical characteristics of low-grade CBT samples and controls used in ROC curve analysis.

Figure 14 shows the results of a ROC curve analysis. Illustration of the increased test accuracy obtained when LTB4DH is used in relationships with HoxAI 3 and IGFBP5.

Figure 15 shows the results of a Li-near BTMs Discriminated Analysis, with and without LTB4DH, for the detection of CBT.

DETAILED DESCRIPTION

Definitions

The term "marker" means a molecule that is quantitatively or qualitatively associated with the presence of a biological phenomenon. Examples of "markers" include a polynucleotide, such as a gene, gene fragment, RNA, or RNA fragment; or a degene product, including a polypeptide such as a peptide, oligopeptide protein or protein fragment; or related metabolites, by products or other identifying molecules, such as antibodies or antibody fragments either directly or indirectly related to a mechanism underlying the phenomenon. Markers of the invention include nucleotide sequences (for example GenBank sequences) as described herein, in particular natural-length sequences, any coding sequences, non-coding sequences and fragments, or any length thereof, and any marker measurable as defined above.

The term "sensitivity" means the proportion of individuals with disease who tests (by the model) positive. Thus, increased sensitivity means lower negative test results.

The term "specificity" means the proportion of individuals with a disease who tests (by the model) negative. Thus, increased specificity means fewer positive test results. The term "expression" includes the production of polynucleotides and polypeptides, in particular the production of RNA (e.g., mRNA) from a gene or portion of a gene, and includes the production of a polypeptide encoded by an RNA or gene or portion of a gene, and includes appearance of a detectable material associated with expression. For example, formation of a complex, for example, of a polypeptide-polypeptide interaction, polypeptide-nucleotide interaction, or the like, is included within the scope of the term "expression". Another example is the binding of a binding ligand, such as a hybridization probe or antibody, to a gene or other polynucleotide, a polypeptide or protein fragment, and the visualization of the binding ligand. Thus, the density of a dot on a microarray, on a hybridization spot such as a northern blot, or on an immunoblot, such as a western blot, or in a bead arrangement, or by PCR analysis, is included within the term " expression "of the biological foundation molecule.

The term "overexpression" is used where the expression of a marker in a cell or cell type is greater than that of another cell or equivalent cell type.

The term "underexpression" is used where the expression of embitterer in a cell or cell type is less than that of another cell or equivalent cell type.

The term "TM" or "tumor marker" or "family memberTM" means a marker that is associated with a particular cancer. Otermo TM also includes combinations of individual markers, whose combination improves the sensitivity and specificity of detecting cancer. It is understood that the term TM does not require the marker to be specific only to a particular tumor. Preferably, TM expression may be altered in other cell types, diseased cells, tumors, including malignant tumors.

TM can be identified by extracting RNA from a tissue sample of a patient suspected of having bladder cancer, applying the RNA or cDNA copy to a microarray having several oligonucleotides in it, allowing the RNA sample to hybridize the oligonucleotides in the array, and in then quantified the assessed RNA level linked to each point of disposition. A marker is considered to be a superexpressant TM if its presence is below a threshold of at least about 1.2-fold that found in normal non-malignant tissue using microarray methods. Alternatively, the threshold may be below about 2 times normal, about 3 times lower than normal, 4 times or about 5 times lower than normal. By "normal" we mean less than 90 percentile of the normal population. In other cases, normal may mean a 95th percentile presence level (ie, about 2 standard deviations (SD) from the mean), and in other cases, less than about 97.5 percent (ie, about 3 SD) or the 99th percentile.

The term "overexpressing TM" means a marker that shows less expression in bladder tumors than in nonmalignant bladder tissue.

The term "overexpressing TM" means a marker that shows greater expression in bladder tumors than in nonmalignant tissue.

The term "TM" or "bladder tumor marker" or "TM family member" means a TM that is associated with bladder cancer. Otermo TM also includes combinations of individual markers, whose combination improves the sensitivity and specificity of detecting bladder cancer. It should be understood that the term TM does not require the marker to be specific to bladder tumors only. Preferably, the expression of deTM may be altered in other cell types, diseased cells, tumors, including malignant tumors.

The term "TM subexpressant" means a marker that shows less expression in bladder tumors than in nonmalignant bladder tissue.

The term "overexpressing TM" means a marker that shows greater expression in bladder tumors than in nonmalignant tissue.

The term "qPCR" means quanitative polymerase chain reaction. The term "qPCR" or "QPCR" refers to the quantitative polymerase chain reaction as described, for example, in PCR Technique: Quantitative PCR, JW Larrick, ed., Eaton Publishing, 1997, and AZ of QuantitativePCR, S. Bustin1 ed., IUL Press, 2004.

The term "CBT" means bladder transitional cell carcinoma. CBTs constitute -95% of all bladder cancers.

As used herein "antibodies" and similar terms refer to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, that is, molecules that contain an antigen binding site that specifically binds (immunoreacts with) an ancient one. These include, but are not limited to, single-stranded, polyclonal, monoclonal, chimeric, Fe, Fab, Fab ', and Fab2 fragments, and a Fab expression library. Antibody molecules refer to any of the classes IgG, IgM, IgA, IgE, and IgD, which differ from each other by the heavy chain nature present in the molecule. These subclasses also include, such as IgGI, IgG2, and others. The light chain may be a kappa chain or a lambda chain. Reference herein to antibodies includes a reference to all classes, subclasses, and types. Also included are chimeric antibodies, for example monoclonal antibodies or fragments thereof which are specific to more than one source, for example, a mouse or human sequence. Also included are camelid antibodies, antibody or shark nanobodies.

The terms "cancer" and "cancerous" refer to or describe physiological condition in mammals that is typically characterized by abnormal or unregulated cell growth. Cancer and cancer pathology may be associated, for example, with metastasis, interference with normal functioning of neighboring cells, release of cytokines or other secreting products at abnormal levels, suppression or aggravation of inflammatory or immunological response, neoplasia, premalignity, malignancy, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc.

The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.

The term "microarray" refers to an orderly or unordered arrangement of capture agents, preferably polynucleotides (e.g. probes) or polypeptides on a substrate. See, for example, Microarray Analysis, M. Schena, John Wiley & Sons, 2002; Microarray Bio-chip Technology, M. Schena1 ed., Eaton Publishing, 2000; Guide to Analysis of DNA Microarray Data, S. Knudsen, John Wiley & Sons, 2004; and protein Microarray Technology, D. Kambhampati, ed., John Wiley & Sons, 2004.

The term "oligonucleotide" refers to a polynucleotide, typically a probe or primer, including, without limitation, single stranded deoxyribonucleotides, single or double stranded ribonucleotides, RNA: DNA hybrids, and double stranded DNAs . Oligonucleotides, such as single stranded DNA probe oligonucleotides, are often synthesized by chemical methods, for example using commercially available automated oligonucleotide synthesizers, or by a variety of other methods, including expression systems. in vitro, recombinant techniques, and expression in cells and organisms.

The term "polynucleotide," when used singularly or plurally, generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. This includes, without limitation, single stranded DNA or double, DNA including single and double stranded regions, single or double stranded RNA, eRNA including single or double stranded regions, hybrid molecules comprising DNA and RNA which may be single stranded or, most commonly, single stranded or include single stranded regions or duo. Also included are triple stranded regions comprising RNA or DNA or both RNA and DNA. Specifically included are mRNAs, cDNAs, and genomic DNAs, and any fragments thereof. The term includes DNAs and RNAs that contain one or more modified bases, such as basalted, or unusual bases, such as inosine. Invention polynucleotides may comprise coding or non-coding sequences, or sequences of sense or antisense. It will be understood that each reference to a "polynucleotide" or similar term, included, will include the naturally-sized sequences as well as any fragments, derivatives, or variants thereof.

"Polypeptide," as used herein, refers to an oligopeptide, peptide, or protein sequence or fragment thereof, and to naturally occurring, recombinant, synthetic, or semi-synthetic molecules. Where "polypeptide" is mentioned herein to refer to an amino acid sequence of a naturally occurring protein molecule, "polypeptide" and similar terms, are not intended to limit the amino acid sequence to native amino acid sequence. , complete, for the naturally-sized molecule. It will be understood that each reference to a "polypeptide" or similar term, included, will include the full-length sequence as well as any fragments, derivatives, or variants thereof.

The "hardness" of hybridization reactions is readily determinable by one of ordinary skill in the art, and is generally an empirical calculation dependent on probe length, wash temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization generally depends on the ability of denatured DNA to anneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative temperatures would tend to make reaction conditions more stringent, while lower temperatures would do less. Additional details and explanation of stringency of hybridization reactions are found, for example, in Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

"Strict conditions" or "high stringency conditions" as defined herein typically: (1) employs low tonic resistance and high wash temperature, for example 0.015 M sodium chloride / 0.0015 M sodium citrate 0.1% sodium dodecyl sulfate at 50 ° C; (2) employ a denaturing agent during hybridization, such as formalin, for example 50% (v / v) formamide with 0.1% bovine wheybumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42 ° C; or (3) employ 50% formamide, 5X SSG (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5X , Denhardt's solution, soundwave-treated sperm DNA (50 pg / ml), 0.1% SDS, and 10% dextran sulfate at 42 ° C, with washes at 42 ° C in 0.2X SSC (chloride sodium / sodium citrate) and 50% formamide at 55 ° C, followed by a high stringency wash comprising 0.1 X SSC containing EDTA at 55 ° C.

"Moderate stringency conditions" can be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of wash solution and hybridization conditions (eg temperature, ionic resistance, and% SDS) less stringent than those described above. An example of moderately stringent conditions is overnight incubation at 37 ° C in a solution comprising: 20% formamide, 5XSSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM desodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 mg / ml denatured sheared salmon sperm DNA, followed by washing 1X SSC filters at about 37 to 50 ° C. The skilled technician will know how to adjust the temperature, ionic resistance, etc. when necessary to accommodate factors such as probe length and the like.

The practice of the present invention will employ, unless otherwise indicated, conventional molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry techniques, which are within the skill of the art. Such techniques are fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd edition, Sambrook et al., 1989; Oligonucleotide Synthesis, MJ Gait, ed., 1984; Animal Cell Culture, R.I. Freshney, ed., 1987; Methods in Enzymology, Academic Press, Inc .; Handbook of Experimental Immunology, 4th edition, D.M. Weir & CC. Blackwell, eds., Blackwell Science Inc., 1987; Gene Transfer Vectors for Mammalian Cells, J.M. Miller & M.P. Callos, eds., 1987; Current Protocols in Molecular Biology, F.M. Ausubel et al., Eds., 1987; and PCR: The Polymerase Chain Reaction, Mullis et al., eds., 1994.

Description of Modes of the Invention

Using a combination of microarray analysis and quantitative polymerase chain reaction (qPCR), markers for transitional cell bladder carcinoma (TCC) that are subexpressed in tumors have been identified. It has been surprisingly found that relationships between these markers and other bladder tumor (TM) 1 markers, especially markers that are overexpressed in tumors, are diagnostic for bladder cancer.

The rates (preferably by assessing an absolute level of a marker) identify a single gene expression "signature" that represents bladder cell cancer, and surprisingly are stronger for variations in urine sampling or concentration techniques.

In addition, the combination of a subexpressed marker and a superexpressed marker maximizes the differential between patient samples and nonmalignant controls, increasing test safety. The underpressure markers described here were selected based on (i) strong and consistent CBT under-regulation, (ii) elevated normal tissue expression, and (iii) insignificant whole-blood expression to minimize the risk of false positives in patients. who have hematuria.

As an alternative for determining the relationship of the two TMs, it has also been found that underexpressed and overexpressed TMs can be analyzed in regression analysis or classification techniques including linear discriminated analysis, and the results of these analyzes are also indicative of a presence of cancer of bladder.

The test involves the evaluation of at least two TM markers, such as a TM, in a sample of a patient suspected of having a cancer or at risk of cancer, where at least one of the TMs is an under-expressed TM. . The rate of underexpressed TM and the other TM is indicative of the presence of cancer. The second TM may be any TM as known in the art, but preferably is an overexpressed TM. Figure 3 shows a number of subexpressed markers suitable for use in the present invention.

The test is best performed using an under-expressed TM in combination with an over-expressed TM. Any overexpressed TM can be used, for example. Identified known overexcess BTMs of invasive bladder tumors (defined herein as> stage1 tumors) are outlined in Figure 11, and identified overexpressed TM of superficial bladder tumors (defined herein as Ta and Tis stage tumors) are shown. in figure 12.

It has also been surprisingly established that preferred sub-expressed BTMs for use in the present invention are those that are not significantly elevated in whole blood, and are present in sufficiently high copy numbers in both tumor cells and nonmalignant bladder cells. Preferred subexpressed BTMs are outlined in Figure 4.

Cancer markers may be detected in a sample using any suitable technique, and may include, but are not limited to, oligonucleotide probes, qPCR, or antibodies constructed against cancer markers.

It will be appreciated that the sample to be tested is not restricted to a sample of tissue suspected to be a tumor. The marker may be secreted into serum, released from cell membranes, released from lysed cells or associated with cells lost in urine. Therefore, a sample may include any body sample, and include biopsies, blood, serum, peritoneal washes, cerebrospinal fluid, urine, and evacuation samples.

It will also be appreciated that the present invention is not restricted to the detection of cancer in humans, but is suitable for the detection of cancer in any animal, including, but not limited to dogs, cats, horses, cattle, sheep, deer, pigs. and any other animals known to contract cancer. General Methods for Cancer Detection

The following methods are non-limiting methods that can be used to evaluate TMs. Following the evaluation of individual TMs, relationships between high and low expression TM family members are determined. These relationships are used to predict the presence or absence of cancer.

Alternatively, high and low expression TMs are used in regression or classification analysis. The results of these analyzes are also used to predict the presence or absence of cancer.

General methodologies for determining expression levels are outlined below, although it is appreciated that any method for determining expression levels is appropriate.

Quantitative PCR (aPCR)

Quantitative PCR (qPCR) can be performed on tumor samples, serum, plasma, and urine samples using TM-specific primers and probes. In controlled reactions, the amount of product formed in a PCR reaction (Sambrook, J., E. Fritsch1 E. and T. Maniatis, Molecular Cloning: A Laboratory Manual 3. Cold Spring Harbor Laboratory Press: Cold Spring Harbor (2001)) correlates with the amount of starting pattern. PCR product quantification can be performed by interrupting the PCR reaction when it is in the log phase, before reagents become limiting. The PCR products are then electrophoresed on agarose or polyacrylamide gels, stained with ethidium bromide or a comparable DNA spot, and staining intensity assessed by densitometry. Alternatively, the progression of a PCR reaction can be assessed using PCR machines such as AppliedBiosystems' Prism 7000 or Roche LightCycIer which assesses product accumulation in real time. Real-time PCR evaluates either DNA interleaving DNA fluorescence such as Sybr Green in the synthesized PCR product, or fluorescence released by a reporter molecule when cleaving a splicing molecule; the reporter and sateer molecules are incorporated into an oligonucleotide probe that hybridizes to the target DNA molecule following the DNA strand extension of the starting oligonucleotides. The oligonucleotide probe is removed and degraded by the enzymatic action of Taq polymerase in the next PCR cycle, releasing the reporter from the sating molecule. In one variation, known as Scorpion®, the probe is covalently linked to the primer.

Reverse Transcription PCR (RT-PCR)

RT-PCR can be used to compare RNA levels in different sample populations in normal and tumor tissues with drug treatment, to characterize expression patterns, to discriminate between closely related RNAs, and to analyze the structured RNA.

For RT-PCR, the first step is RNA isolation from a target sample. The starting material is typically total RNA isolated from human tumors or tumor cell lines, and corresponding to normal tissues or cell lines, respectively. RNA can be isolated from a variety of samples, such as breast, lung, hand, colon (e.g., large or small intestine), colorectal, gastric, esophageal, anal, rectal, prostate, brain, liver, kidney, pancreas, spleen, thymus, testis, ovary, uterus, bladder, etc., tissues, primary tumors, or tumor cell lines, and collected samples from healthy donors. If the source of RNA is a tumor, the RNA can be extracted, for example, from frozen and fi xed (fi xed formalin fixed) paraffin embedded or paraffin embedded tissue samples.

The first step in RT-PCR gene expression profiling is reverse transcription of the RNA pattern into cDNA, followed by its exponential amplification in a PCR reaction. The two most commonly used reverse transcriptases are avian myeloblast virus reverse transcriptase (AMV-RT) and murine leukemia murine leukemia reverse transcriptase (MMLV-RT). The reverse transcription step is typically prepared using specific primers, random hexamers, or oligo-dT primers, depending on the circumstances and the purpose of the expression profile. For example, extracted RNA may be transcribed inversely using a geneAmp RNA PCR Kit (Perkin Elmer, CA, USA) following the manufacturer's instructions. Derived cDNA can then be used as a standard in the subsequent PCR reaction.

Although the PCR step may be used, a variety of thermostable DNA dependent DNA polymerases, it typically employs aTaq DNA polymerase, which has a 5'-3 'nuclease activity but has no 3'-5' review endonuclease activity. Thus, TaqMan (q) PCR typically uses the 5 'nuclease activity of Taq or Tth polymerase to hybridize a hybridization probe bound to its target amplicon, but any equivalent 5' activity enzyme can be used.

Two oligonucleotide primers are used to generate a typicalplicon of a PCR reaction. A third oligonucleotide, or probe, is designed to detect nucleotide sequence located between the two PCR primers. The probe is not extendible by Taq DNA polymerase enzyme, and is labeled with a reporter fluorescent dye and a fluorescent dye dye. Any laser-induced emission of the dye reporter is sasciated by the sating dye when the two dyes are located next to each other when they are over the probe. During the amplification reaction, the Taq DNA polymerase enzyme cleaves the probe in a standard-dependent manner. The resulting probe fragments dissociate in solution, and the signal from the released tincture of the reporter is free from the satiety effect of the second fluorophor. One dye-reporter molecule is released for each new synthesized molecule, and detection of the unsaturated dye-reporter provides the basis for quantitative data interpretation.

TaqMan RT-PCR can be performed using commercially available equipment such as, for example, ABI PRISM 7700 Sequence Detection System (Perkin-Elmer-Applied Biosystems, Foster City, CA, USA), or Lightcycler (Roche Molecular Biochemicals, Mannheim, Germany). In a preferred embodiment, the 5 'nuclease procedure is performed on a quantitative real-time PCR device such as the ABI PRISM 7700tam Sequence Detection System. The system consists of a thermal cycler, laser, charge coupled device (CCD) 1 camera, and computer. The system amplifies samples in a 96-well format without a thermal cycler. During amplification, the laser-induced fluorescent signal is collected in real time using 96-well optical fiber cables and detected as CCD. The system includes software to operate the instrument and to analyze the data.

5 'nuclease assay data are initially expressed as Ct, or the borderline cycle. As described above, fluorescence values are recorded during each cycle and represent the amount of product amplified for that point in the amplification reaction. The point when the fluorescent signal is first recorded as statistically significant is the borderline cycle.

Real-Time Quantitative PCR (aPCR)

A more recent variation of the RT-PCR technique is quantitative real-time PCR, which assesses the accumulation of PCR product by means of a dual labeled fluorigenic probe (ie TaqMan probe). Real-time PCR is compatible with both quantitative competitive PCR and quantitative comparative PCR. The former uses an internal competitor for each target sequence for normalization, while the latter utilizes a contiguous normalization gene within a sample, or an RT-PCR maintenance gene. Further details are provided, for example, by Held et al., Genome Research 6: 986-994 (1996).

Expression levels can be determined using fixed paraffin embedded tissues as the RNA source. According to a common aspect of the present invention, PCR primers and probes are defined based on the intron sequences present in the gene to be amplified. In this mode, the first step in the primer / probe design is the design of intron sequences within the genes. This may be done by publicly available software, such as DNA BLAT software developed by Kent, W. J., Genome Res. 12 (4): 656-64 (2002), or by BLAST software including variations thereof. Subsequent steps follow well-established PCR primer and probe design methods. In order to avoid nonspecific signals, it is useful to mask repetitive sequences within the introns when designating primers and probes. This can be easily accomplished using the Repeat Masker program available online. line through Baylor College of Medicine, which evaluates DNA sequences against a library of repetitive elements and returns an inquiry sequence in which repetitive elements are masked. Masked sequences can then be used to designate primer and probe sequences using any commercially or otherwise publicly available primer / probe planning packages, such as Primer Express (Applied Biosystems); MGB assay-by-design (Applied Biosystems); Primer3 (Steve Rozen and Helen J. Skaletsky (2000) Primer3 on the WWW for general users and programmersbiologists at: Krawetz S, Misener S (eds) Bioinformatics Methods and Pro-tocols: Methods in Molecular Biology. Human Press, Totowa, NJ, pp 365-386).

The most important factors considered in PCR primer design include primer length, melting temperature (Tm), G / C eteor, specificity, complementary primers, and 3 'end sequence. In general, ideal PCR primers are generally 17 to 30 bases in length, and contain about 20 to 80%, such as, for example, about 50 to 60% G + C bases. Melting temperatures between 50 and 80 ° C, for example about 50 to 70 ° C, are typically preferred. For other standards for the PCR primer and see-probe design, for example, Dieffenbach, CW and others, General Concepts for PCR Prime Design at: PCR Primer, A Laboratory Manual, Cold Spring Harbor La-boratory Press, New York, 1995, pp. 133-155; Innis and Gelfand, Optimizati-on of PCRs in: PCR Protocols, A Guide to Methods and Applications, CRCPress, London, 1994, p. 5-11; and Plasterer, Τ. N. Primerselect: Primer andprobe design. Methods Mol. Biol. 70: 520-527 (1997), the entire descriptions of which are hereby expressly incorporated by reference.

Microarray Analysis

Differential expression may also be identified, or confirmed using the microarray technique. In this way, the CCPM expression profile can be evaluated in fresh or paraffin embedded tumor tissue using microarray technology. In this method, polynucleotide sequences of interest (including cDNAs and oligonucleotides) are seeded, or arranged, on a microchip substrate. The arranged sequences (i.e. capture probes) are then hybridized to cell or tissue-specific compolinucleotides of interest (i.e. aliens). Just as in the RT-PCR method, the RNA source is typically total RNA isolated from human tumors or tumor cell lines, and corresponding normal tissues or cell lines. In this way the RNA can be isolated from a variety of primary tumors or tumor cell lines. If the RNA source is a primary tumor, RNA may be extracted, for example, from frozen formalin-fixed paraffin-embedded tissue (FFPE) tissue samples and fixed-tissue (eg formalin-fixed) tissue samples, which are routinely prepared. -rade and preserved in clinical practice every day.

In a specific embodiment of the microarray technique, PCR amplified insertions of cDNA clones are applied to a substrate. The substrate may include up to 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 75 nucleotide sequences. In other aspects, the substrate may include at least 10,000 nucleotide sequences. The microarrayed sequences immobilized on the microchip are suitable for hybridization under stringent conditions. As other embodiments, targets for microarray may be at least 50, 100, 200, 400, 500, 1000, or 2000 bases in length; or 50-100, 100-200, 100-500, 100-1000, 100-2000, or 500-5000 bases in length. As other embodiments, the microarray capture probes may be at least 10, 15, 20, 25.50, 75, 80, or 100 bases in length; or 10-15, 10-20, 10-25, 10-50,10-75, 10-80, or 20-80 bases in length.

Fluorescently labeled cDNA probes can be generated by incorporating reverse transcriptional fluorescent nucleotides from RNA extracted from tissues of interest. Rotated cDNA probes applied to the chip hybridize with specificity for each of the arrayed DNA points. After rigorous washing to remove non-specifically bound probes, the chip is evaluated by laser microscopy confocal or by another detection method, such as a CCD camera. Hybridization quantitation of each arrayed element provides the corresponding mRNA abundance assessment. With dual color fluorescence, separately labeled cDNA probes generated from two RNA sources are paired for arrangement. The relative abundance of transcripts from the two sources corresponding to each specified gene is determined simultaneously. An exemplary protocol for this is described in detail in Example 4.

The miniaturized hybridization scale provides a convenient and rapid assessment of the expression pattern for large numbers of genes. Such methods have been shown to have the sensitivity required to detect rare transcripts, which are expressed in some copies by cell, and to reproducibly detect at least approximately two-fold differences in expression level (Schena et al., Proc. Natl. A -cad Sci USA 93 (2): 106-149 (1996)). Microarray analysis can be performed by commercially available equipment following manufacturer's protocols, such as using Affymetrix GenChip technology, Illumina microarray technology or Incyte microarray technology.

The development of microarray methods for large-scale gene expression analysis makes it possible to systematically search for molecular markers of cancer classification and outcome prediction in a variety of tumor types.

Isolation. RNA Purification, and Amplification

General methods for mRNA extraction are well known in the art and are described in standard molecular biology textbooks, including Ausubel et al., Current Protocols of Molecular Biology, John Wileye Sons (1997). Methods for RNA extraction from emparaffin embedded tissues are described, for example, in Rupp and Locker, Lab Invest. 56: A67 (1987), and De Sandres et al., BioTechniques 18: 42044 (1995). In particular, RNA isolation can be performed using purification kit, buffer group, and protease from commercial manufacturers such as Qiagen according to the manufacturer's instructions. For example, total cell RNA in culture can be isolated using Qiagen RNeasy mini columns. Other commercially available RNA isolation kits include MasterPure Complete DNA and RNA Purification Kit (EPICENTRE (D, Madison, Wl), and Paraffin Block RNA Isolation Kit (Ambion, Inc.). tissue can be isolated using Stat-60 RNA (Tel-Test) Tumor prepared RNA can be isolated, for example, by cesium chloride density gradient centrifugation.

The steps of a representative protocol for gene expression profiling using fixed paraffin-embedded tissues such as RNA source, including mRNA isolation, purification, primer extension, and amplification are provided in various published journal articles (eg. Example: E. E. Godfrey et al., J. Molec. Diagnostics 2: 84-91 (2000); K.Specht et al., Am. J. Pathol. 158: 419-29 (2001)). Briefly, a representative process begins with cutting about 10 µm thick desections of embedded paraffin tumor tissue samples. RNA is then extracted, and protein and DNA are removed. After RNA concentration analysis, RNA repair and / or amplification steps may be included if necessary, and RNA is transcribed backwards using specific gene promoters followed by RT-PCR. Finally, data are analyzed to identify the best available treatment options for the opacient based on the characteristic gene expression pattern identified in the tumor sample examined.

Immunohistochemistry and Proteomics

Immunohistochemical methods are also suitable for detecting the expression levels of proliferation markers of the present invention. In this manner, antibodies or antisera, preferably polyclonal antisera, and more preferably marker specific monoclonal antibodies, are used to detect expression. Antibodies can be detected by direct labeling of the antibodies themselves, for example with radioactive labels, fluorescent labels, haphenene labels such as biotin, or an enzyme such as horseradish peroxidase or alkaline phosphatase. Alternatively, unlabelled primary antibody is used in conjunction with a labeled secondary antibody comprising antisera, polyclonal antisera or a monoclonal antibody specific for the primary antibody. Immunohistochemistry kits and protocols are well known in the art and are commercially available.

Proteomics can be used to analyze the polypeptides present in a sample (eg, tissue, organism, or cell culture) at a certain point in time. In particular, proteomic techniques may be used to estimate overall changes in polypeptide expression in a sample (also referred to as expression proteomics).

Proteomic analysis typically includes: (1) separation of individual polypeptides into a sample by 2-D PAGE gel electrophoresis; (2) identification of the individual polypeptides recovered from the gel, for example by mass spectrometry or N-terminal sequencing, and (3) data analysis using bioinformatics. Proteomic methods are valuable supplements to other gene expression profiling methods, and may be used alone or in combination with other methods to detect the proliferation marker products of the present invention.

Hybridization Methods Using Selective Nucleic Acid Probes for a Label

These methods involve binding the nucleic acid probe to a support, and hybridization under appropriate conditions with RNA or cDNA derived from the test sample (Sambrook, J., Fritsch, E. and T. Maniatis, Molecular Cloning: A Laboratory Manual 3â. Cold Spring Harbor LaboratoryPress: Cold Spring Harbor (2001)). These methods can be applied to TM derived from a tumor tissue or fluid sample. RNA or cDNA preparations are typically labeled with a fluorescent or radioactive molecule to allow detection and quantitation. In some applications, DNA hybridization may be labeled with a fluorescently labeled structure branched to enhance signal intensity (Night, FS, Branched DNA signal amplification for direct quantitation of nucleic acid sequences in clinical specimens. Adv. Clin Chem 33, 201-35 (1998)). Unhybridized label is removed by extensive washing in salt solutions such as 0.1 χ SSC, 0.5% SDS before quantifying the amount of hybridization by fluorescence detection or gel image densitometry. The supports may be solid, such as nylon or nitrocellulose membranes, or consist of microspheres or beads that are hybridized when in liquid suspension. To allow washing and purification, the beads may be magnetic (Haukanes, BI and Kvam, C., Application of magnetic beads in bioassays. Bio / Technology 11, 60-63 (1993)) or fluorescently labeled for allow flow cytometry (see for example: Spiro, A., Lowe, M. and Brown, D., A Bead-Based Method for Multiplexed Identification and Quantitation of DNA Sequences Using Flow Cytometry. Appl. Env. Mi-cro. 66, 4258-4265 (2000)).

A variation of hybridization technology is the Quanti-Gene Plex® Assay (Genospectra, Fremont) which combines a fluorescent bead support with branched DNA signal amplification. Still another variation in hybridization technology is the Quantikine® mRNA assay (R&D Sys-tems, Minneapolis). The methodology is as described in the manufacturer's instructions. Briefly the assay uses Digoxigenin-conjugated oligonucleotide hybridization probes. Hybridization is detected using alkaline phosphatase-coupled anti-Digoxigenin antibodies in colorimetric assays.

Additional methods are well known in the art and need not be described here as well.

Enzyme-Linked Immunoassay Assays (ELISA)

Briefly, in sandwich ELISA assays, an anti-polyclonal or monoclonal against TM is attached to a solid support (Crow-ther, JR The ELISA guidebook. Human Press: New Jersey (2000); Harlow, E. and Lane, D. using antibodies: a laboratory manual Cold S-pring Harbor Laboratory Press: Cold Spring Harbor (1999)) or suspending beads. Other methods are known in the art and need not be described herein either. Monoclonal antibodies may be derived from hybridoma or selected from phage antibody libraries (Hust M. and Dubel S., Phage display vectors for the in vitro generation of human fragments of antibody. Metods Mol Biol. 295: 71-96 (2005) ). Non-specific binding sites are blocked with non-target protein preparations and detergents. The capture antibody is then incubated with a urine or tissue preparation containing the TM antigen. The mixture is washed before the antibody / antigen complex is incubated with a second antibody that detects the target TM. The second antibody is typically conjugated to a fluorescent molecule or other reporter molecule that can be detected in an enzyme reaction or with a third antibody conjugated to a reporter (Crowther, Idi). Alternatively, in direct ELISAs, the preparation containing the TM may be bound to the support or bead and the target antigen detected directly with an antibody-reporter conjugate (Crowther, Id.).

Methods for producing monoclonal and antisera-polyclonal antibodies are well known in the art and need not be described here either.

Immunodetection

The methods may also be used to immunodetect marker family members in serum or plasma from bladder cancer patients harvested before and after surgery to remove the tumor, immunodetection of marker family members in patients with other but not limited to cancers. a, colorectal, pancreatic, ovarian, melanoma, liver, esophageal, stomach, endometrial, and cerebral and immunodetection of marker family members in urine and evacuation of bladder cancer patients.

BTMs can also be detected in tissues or urine using other standard immunodetection techniques such as immunoblotting or immunoprecipitation (Harlow, E. and Lane, Di using antibodies: a laboratory manual. Cold Spring Harbor Laboratory Press: Cold Spring Harbor) (1999)). In immunoblotting, tissue or fluid protein preparations containing the TM are electrophoresed through polyacrylamide gels under denaturing or non-denaturing conditions. The proteins are then transferred on a membrane support such as nylon. TM is then reacted directly or indirectly with monoclonal or polyclonal antibodies as described for immunohistochemistry. Alternatively, in some preparations, proteins may be stained directly on membranes without prior electrophoretic separation. The signal can be quantified by densitometry.

In immunoprecipitation, a soluble preparation containing TM is incubated with a monoclonal or polyclonal antibody against TM. The reaction is then incubated with inert beads made of agarose or polyacrylated with covalently bound protein G or protein A. Protein A or G beads specifically interact with the antibodies forming an immobilized antibody-TM-antigen bound complex to the bead. Following leverage, the bound TM can be detected and quantified by immunoblotting or ELISA.

Threshold Determination

For tests using sub-regulated BTMs in detax or regression analysis, thresholds will be derived that will allow a sample to be called positive or negative for CBT. These thresholds will be determined by analyzing cohorts of patients being investigated for the presence of CBT. Thresholds may vary for different test applications; for example, thresholds for test use in population assessment will be determined using cohorts of patients who are largely free of urological symptoms, and these thresholds may differ from those used in tests for patients under surveillance for TCC. , or those being investigated for the presence of urologic disintomas such as hematuria. A threshold can be selected to provide a practical level of test specificity in the required clinical scenario; that is, the specificity that allows reasonable sensitivity without excessive numbers of patients receiving false positive results.

This specificity may be within the range of 80-90%. An alternative method for obtaining a test threshold is to plot sensitivity against specificity for different test thresholds (ROC curves) and then select the curve's inflection point.

As an alternative to single thresholds, the test may use test ranges that provide different degrees of likelihood of disease presence and have different clinical consequences associated with them. For example, a test may have three intervals; an associated common high risk (eg 90%) of the presence of CBT, a second associated with a low risk of CBT and a third considered to be a suspected disease. The "suspicious" interval may be associated with a recommandation for a repeat test over a defined period of time.

Methods to Detect Urine Bladder Cancer Markers

In various embodiments, assays for TM may desirably be performed on urine samples. In general, assay methods for oligonucleotides, proteins and peptides in these fluids are known in the art. However, for illustration purposes, levels of urine from a TM can be quantified using a sandwich enzyme-linked immunosorbent assay (ELISA). For plasma or serum assays, a 5 μL aliquot of an appropriately diluted sample or TM consecutively diluted and 75 μί. of peroxidase-conjugated anti-human TM antibody are added to the wells of a microtiter plate. After a 30 minute incubation period at 30 ° C, the wells are washed with 0.05% Tween 20 in phosphate buffered saline (PBS) to remove unbound antibody. Bound TM complexes and anti-TM antibody are then incubated with o-phenylendiamine containing H2 O2 for 15 minutes at 30 ° C. The reaction is stopped by the addition of 1 M H2SO4, and absorbance at 492 nm is measured with a microtiter placard reader. It may be appreciated that anti-TM antibodies may be monoclonal antibodies or polyclonal antisera.

Because many proteins are (1) secreted by cells, (2) cleaved from cell membranes, (3) cells lost from cell death or (4) contained within loose cells, it will be appreciated that BTMs can also be detected. in the urine. Additionally, bladder cancer diagnosis can be determined by measuring BTM expression in a sample, or BTM accumulation in a sample. Prior art diagnostic methods include cystoscopy, cytology, and examination of cells extracted during these procedures. Such methods rely on the identification of tumor cells in the urine or in a urothelial brush sample, or in other cases in bladder wall biopsy specimens. These methods suffer from various types of errors, including sampling error, observer identification errors, and the like.

Antibodies to Bladder Tumor Markers

In additional aspects, this invention includes manufacture of antibodies against BTMs. Using methods described herein, new BTMs can be identified using microarray and / or qPCR methods. Once a putative marker is identified, it can be produced in sufficient quantities to be adequate to elicit an immune response. In some cases a life-size TM may be used, and in others a peptide fragment of a TM may be sufficient as an immunogen. The immunogen may be injected into a suitable host (e.g., mouse-dongo, rabbit, etc.) and if desired, an adjuvant such as Freund's complete adjuvant, incomplete Freund's adjuvant may be injected to increase immune response. It may be appreciated that antibody preparation is routine in immunological techniques and need not be described herein either. As a result, one can produce antibodies against identified BTMs using methods described herein.

In still other embodiments, antibodies may be prepared against the protein or protein nucleus of the tumor markers identified herein or against an oligonucleotide sequence unmatched by a TM. Although certain proteins may be glycosylated, variations in the glycosylation pattern may, under certain circumstances, result in poor detection of forms of BTMs lacking the usual glycosylation patterns. Thus, in certain aspects of this invention, TM immunogens may include deglycosylated TM or deglycosylated TM fragments. Deglycosylation may be performed using one or more glycosidases known in the art. Alternatively, TM cDNA may be expressed in glycosylation-deficient cell lines, such as prokaryotic cell lines, including E. coli and the like.

Vectors may be prepared by having TM coding oligonucleotides in them. Many of such vectors may be based on standard vectors known in the art. Vectors can be used to transfect a variety of cell lines to produce TM1-producing cell lines that can be used to produce desired amounts of TM for the development of specific antibodies or other reagents for BTM detection or standardization of assay. developed for BTMs.

Kits

Based on the findings of this invention, various types of test kits can be considered and produced. First, kits that can be prepared have a detection device preloaded with a detection molecule (or "capture reagent"). In mRNA detection modalities, such devices may comprise a substrate (e.g., glass, silicon, quartz, metal, etc.) on which oligonucleotides as capture reagents that hybridize to the mRNA to be de- ceilings are connected. In some embodiments, direct detection of mRNA may be performed by mRNA hybridization (labeled cy3, cy5, radiolabelled or other label) to the oligonucleotides on the substrate. In other embodiments, mRNA detection may be performed first by preparing complementary DNA (cDNA) to the desired mRNA. Then, labeled cDNA can be hybridized to oligonucleotides on the substrate and detected.

Antibodies may also be used in kits as capture reagents. In some embodiments, a substrate (e.g., a multiwell plate) may have a specific TM capture reagent attached thereto. In some embodiments, a kit may have a blocking reagent included. Blocking reagents may be used to reduce non-specific binding. For example, non-specific oligonucleotide binding can be reduced by using excess DNA from any convenient source that does not contain TM oligonucleotides, such as salmon sperm DNA. Nonspecific antibody binding can be reduced by using an excess of a blocking protein such as dehydrating albumin. It may be appreciated that numerous methods for detecting oligonucleotides and proteins are known in the art, and any strategy that can specifically detect TM-associated molecules may be employed and considered within the scope of this invention.

In addition to a substrate, a test kit may comprise capture reagents (such as probes), wash solutions (e.g. SSC, other salts, buffers, detergents and the like) as well as detecting portions (e.g. cy3). , cy5, radiolabelled, and the like). Kits may also include instructions for use and a pack. TM rates used for bladder cancer detection I

In a number of embodiments, reagents for TMLTBDH4 testing in combination with overexpressing BTMs may be incorporated into a kit for testing unfractionated urine or urine cell sediment to detect bladder cancer. Urine specimens may be collected from patients with diagnosed bladder cancer who require monitoring for disease progression or treatment response, individuals with urological symptoms including macroscopic or microscopic hematuria, or asymptomatic individuals. For patients or individuals being tested with a kit that measures BTMs in unfractionated urine, approximately 2 ml of urine may be collected for testing. For testing on the urine pellet,> 10 ml of urine may be collected.

A suitable kit includes: (i) instructions for use and result interpretation, (ii) software for interpretation of multiple gene analyzes, including any regression analysis classifier or reagents of formula (iii) for stabilization and purification of non-urine RNA fractionated or urine pellets, (iv) cDNA synthesis reagents including NTPs and reverse transcriptase, and (v) reagents for TM cD-NA quantitation. In one form, these reagents would be used for quantitative PCR and would include oligonucleotide primers spanning specific exons, a third oligonucleotide labeled with a detection probe, Taq polymerase and the other buffers, salts and dNTPs required for PCR. The kit may also utilize other methods for detecting transcripts such as direct hybridization of TM RNA to probes labeled with branched DNA technology; and (vi) oligonucleotides and probe for detecting transcripts of a highly transcribed gene, such as Bactin, to serve as a quality control measure.

Evaluation of Bladder Cancer Progression Using TM Rates

To assess the progression of bladder tumors, detained specimens are obtained by bladder wall biopsy or urine samples collected over time from a patient having bladder cancer. BTM rate assessment or combinations of these are made for samples collected at different times. TM rates within a specified range are indicative of bladder cancer progression.

Evaluation of Bladder Cancer Therapy Using TM Rates

To evaluate the efficacy of therapy for bladder tumors, tissue and / or urine samples are obtained before treatment is started. Reference levels of one or more BTMs are determined, as are rates of various BTMs with respect to each other. Treatment is initiated, and may include any therapy known in the art, including surgery, radiation therapy, or chemotherapy as appropriate to the type and stage of the disease. During the course of therapy, tissue and / or urine samples are collected and analyzed for the presence and amount of BTMs. Rates of various BTMs are determined and results are compared to: (1) pre-treatment patient referral levels or (2) normal values obtained from a population of individuals who do not have bladder cancer.

Using TM Rates to Monitor CBT Therapy Progression

In addition to rapid diagnosis and early detection of CBT, TM marker rates detected in tissue, serum or urine may be used to monitor a patient's response to therapy. In these applications, samples of urine and / or serum may be collected at intervals following initiation of systemic, intravesicular or intravascular chemotherapy, radiotherapy or immunotherapy. A change in marker rate may indicate a reduction in tumor size, indicative of effective treatment. The change rate can be used to predict the optimal therapeutic dose for each patient or treatment.

Using TM Regression Analyzes

In addition to TM1 rates regression or classification analyzes which include members of the low and high expression TM family may be used for the applications described above.

Evaluated markers are selected from known human genes. The genes evaluated are shown in Figures 3 and 4. Included in Figures 3 and 4 are the gene names, HUGO identifier, MWG right-number, NCBI mRNA reference sequence number, and protein reference number. Natural length sequences can be found at http://www.ncbi.nlm.nih.gov/entrez/.

EXAMPLES

The examples described herein are for purposes of illustrative embodiments of the invention and are not intended to limit the scope of the invention. Other embodiments, methods and types of analysis fall within the scope of persons skilled in molecular diagnostic techniques and need not be described in detail about. of this. Other embodiments within the scope of the art which are based on the teachings herein are considered to be part of this invention.

Methods

Tumor Collection

Bladder tumor samples and non-malignant urothelium samples were collected from resected surgical specimens at KyotoUniversity Hospital, Japan.

Urine Collection

Urine samples from non-malignant controls and bladder cancer patients were obtained from Kyoto University Hospital, Japan. Healthy control samples were obtained from Japanese volunteers (Figure 1).

Extraction of tumor TNATs were homogenized in a mixture of TriReagent: water (3: 1), then extracted chloroform. Total RNA was then purified from the aqueous phase using the RNeasy ™ procedure (Qiagen). RNA was also extracted from 16 knockout cancer cell lines to serve as a reference RNA.

RNA was extracted from the urine by mixing the common urine sample with equal volume of Iise buffer (5.64 M guanidine-HCI, 0.5% sarcosyl, 50 mM sodium acetate (pH 6.5) and 1 mM β-mercaptoethanol (pH adjusted to 7.0 with 1.5 M Hepes pH 8). Due to the low amounts of RNA in the urine, 7.5 µg of total bacterial RNA was added to the urine mixture / lysis buffer to act as a vehicle. Total RNA was then extracted using Trizol and the RNeasy ™ procedure. RNA preparations were also purified prior to cDNA synthesis using the Qiagen QIAquick ™ PCR purification kit.

RNA was extracted from the blood of three healthy volunteers by performing Trizol / RNeasy ™ extraction on bloodlet-enriched cells using 3.6% dextran sedimentation.

Microarray Blade Preparation

Epoxy coated glass slides (MWG Biotech) were labeled with -30,000, 50 mer oligonucleotides (MWG Biotech) using a Gene Machine microarray robot, according to the manufacturer's protocol.

RNA Labeling and Hybridization

cDNA was transcribed from 5 pg of total RNA using Superscript II ™ reverse transcriptase (Invitrogen) in reactions containing 5- (3-aminoallyl) -2 'deoxyuridine-5'-triphosphate. The reaction was then deionized on a Microcon column before being incubated with Cy3 or Cy5 in debicarbonate buffer for 1 hour at room temperature. Unincorporated dyes were removed using a Qiaquick column (Qiagen) and sample concentrated at 15 μl in a SpeedVac. Cy3 and Cy5 labeled cDNAs were then mixed with Ambion ULTRAhyb ™ buffer, denatured at 100 ° C for 2 minutes and hybridized to the microarray slides in the hybridization chambers at 42 ° C for 16 hours. The slides were then washed and analyzed twice on an Axon 4000A ™ scanner at two force settings.

Microarray Analysis of Cancer Marker Genes

RNA from 53 bladder tumors and 20 non-malignant ("normal") bladder tissue samples were labeled with Cy5 and hybridized to duplicates or triplicates with reference RNA labeled by Cy3. After normalization, the change in expression in each of the 29,718 genes was then estimated by doubling change and statistical probability.

Standardization Procedure

The mean fluorescence intensities detected by Genepix ™ softwa-re were corrected by subtracting the local base intensities.

Spots with a corrected base intensity of less than zero were excluded. To facilitate normalization, total spot intensity and intensity rates were log-transformed. Iog intensity rates were corrected for tincture and spatial tendency using local regression implemented in the LOCFIT ™ package. Intensity rates in Yog were regressed simultaneously with respect to total spot intensity and spot. Residues of local regression provided corrected recorded deduplication changes. For quality control, the rates of normalized microarray were plotted with respect to the intensity and localization of the spot. The spots were subsequently visually inspected for any remaining artifacts. Additionally, an ANOVA model was applied for pinpoint trend detection. All normalization results and parameters were entered into a Postgres database for statistical analysis.

Statistical analysis

To improve the comparison of measured duplication changes between layouts, log2 (rates) were scaled to have the same total standard shift per layoff. This standardization reduced class variability in the middle tissue. A classification test based on doubling changes was then used to improve signal strength. This test consists of two steps: (i) calculating the deduplication change (Rfc) classification in the provisions and ii) subtracting the mean (Rfc) to normal tissue (Rfc) for tumor tissue. The difference between both mean scores defines the score for the doubling change rating. Three additional statistical tests were also performed on the standardized data: 1) Two Student's t-test samples, 2) Wilco-xoη test and 3) Microarray statistical analysis (SAM). The most significantly underregulated genes determined by each of the statistical methods (change in classification doubling, t-test, Wilcoxon test, and SAM) were determined in a classification score for each test. All classification scores were then added to a summed classification score.

Urine RNA cDNA Synthesis

Total urine RNA was annealed to degene specific primers for each of the bladder tumor markers by incubating at 70 ° C and then cooling on ice for 2 minutes in 50 µl of reactions containing forward primers at 0.01 Mg / μl. Each cDNA reaction contained annealedRNA and 4 μΙ 5x Superscript Il reverse transcriptase buffer (Invitrogen, USA), 2 μΙ 0.1 M DTT (Invitrogen, USA), 0.5 μΙ RNase (40υ / μL), (Invitrogen, USA), 4 μl of 10 mM dNTP (Invitrogen, USA) and 0.5μl Superscript Il (200υ / μl) reverse transcriptase, (Invitrogen, USA) in a final volume of 20 μΙ. Reactions were incubated at 42 ° C for 1 hour, 10 minutes at 70 ° C and 1 minute at 80 ° C. Reactions were cleared prior to qPCR with Qiagen QIAquick PCR Purification Columns (Qiagen, Victoria, Aus-tralia) and stored at -80 ° C. Quantitative Real-Time PCR

Real-time or quantitative PCR (qPCR) is used for absolute or relative quantification of PCR model copy number. Taqman ™ primer and probe groups were designated using the Express V 2.0 ™ Primer (Applied Biosystems). Where possible, all potential binding variants were included in the resulting amplicon, with amplicon preference given to regions covered by the MWG-Biotech-derived microarray oligonucleotide. The probe and primer sequences are shown in Figure 2. Alternatively, if the target gene was represented by an Assay-on-Demand ™ expression assay (Applied Biosystems) covering the desired amplicons, these were used. In the household designated assays, primer concentration was titrated using a SYBR green labeling protocol and cDNA made from reference RNA. Amplification was performed on an ABIPrism ™ 7000 sequence detection system under standard cycling conditions. When single amplification products were observed in the dissociation curves, the standard curves were generated in a concentration range of 625 doublings using the optimal primer and 5'FAM - 3'TAMRAphosphate Taqman ™ (ProIigo) probe concentrations at a final concentration of 250 nM. Tests determining standard curves with regression coefficients above 0.98 were used in subsequent analyzes.

Assays were performed in 96-well plates. Each plate contained a standard reference cDNA curve within a 625-fold concentration range. For urine qPCR, total RNA extracted from ~ 0.5mls of unfractionated urine was used in each reaction. The ACt (mean reference Ct - cDNA target gene Ct) was calculated for each marker, and used for subsequent rates, regression analysis or classification.

Expression of blood markers

The expression of the markers shown in figures 3 and 4 and all blood was determined in silico. Microarray probes were attached to the UniGene beams through GenBank accession numbers from their target mRNAs, and the UniGene tissue expression profile used to determine the number of sequence labels (ESTs) in blood libraries. Only genes with 0 or 1 expressed sequence labels (EST) are shown in Figure 4. To confirm the low expression of LTB4DH in all blood, RT-qPCR was performed on whole blood extracted RNA using primers and probes. shown in figure 2.

No significant expression was observed observed (results not shown).

Identification of underreacted bladder cancer markers

To identify the downregulated bladder cancer markers, RNA microarray studies were performed on 53 bladder tumors and 20 malignant bladder tissue samples using 30,000 oligonucleotide chips. Figure 3 shows statistical analysis of microarray data for 300 genes showing significant down-regulation in bladder cancer tissue compared to nonmalignant tissue. Figure 3 includes the HUGO gene name and symbol, the protein reference sequence number, the cnNC mRNA reference sequence number, the MWG Biotech probe oligonucleotide number, the average deduplication change in gene expression between the tissue. tumor and nonmalignant, the results of an original unadjusted Student's t-test, the results of the 2-sample Wilcoxon test, the results of the SAM test, and the summed classification score.

Identification of Preferred Sub-Expressed Bladder Tumor Markers for Use in Bladder Cancer Urine Tests

Because urinary hematuria is a common co-occurrence with bladder cancer, it is an advantage that bladder cancer markers are significantly elevated in whole blood. In addition, because down-regulated markers are being used in rates, regression analysis or classification, it is an advantage that they are present in sufficiently high copy numbers in both tumor cells and non-malignant bladder cells to allow reliable detection. in the urine. To identify suitable markers, the genes in figure 3 were evaluated for a subgroup that had little or no representation in the blood EST libraries, and was higher than the average expression in nonmalignant tissue. The mean expression was estimated by classifying the 30,000 oligonucleotides in disposition by their mean intensity in a sample analyzed in the microarray study. Markers meeting the criteria are shown in Figure 4. Figure 4 includes the HUGO gene symbol name, protein reference sequence number, NCBI1 mRNA reference sequence number, mean doubling change, score of classification, the mean intensity classification of microarray stain in tumor tissue and nonmalignant tissue, and the number of ESTs present in blood EST libraries.

The down-regulation observed in the array data was validated by qPCR for three genes shown in Figure 4, LTB4DH, BAG1 and FLJ21511. These genes were tested total RNA from 10 tumor samples and 10 nonmalignant samples. LTB4DH, BAG1 and FLJ21511 showed a mean down-regulation in bladder tumors compared to non-malignant bladder tissue of 2.5 doublings, 1.4 doublings and 6.1 doublings, respectively, in these samples.

Urine qPCR analysis using LTB4DH

The urine of CBT patients and controls with nonmalignant urological conditions was collected either by emptying or by catheterization. Total RNA was extracted from the empty urine of 42 controls and the empty or catheterized urine of 37 CBT patients and used in quantitative RT-PCR using LTB4DH primers and probes and three overexpressed markers, IGFBP5, MDK and HoxAI 3. As ACt rates were determined for IGFBP5 / LTB4DH, MDK / LTB4DH and HoxAI3 / LTB4DH.

This data is illustrated by the box plots in Figure 5, which shows a clear difference in data propagation between control urine samples and CBT patients for each of the three tests. The most accurate test was IGFBP5 / LTB4DH which demonstrated sensitivity and specificity of 87% and 88% in this sample cohort, respectively (Fig. 6a). To illustrate the correlation between sensitivity and specificity for each of these tests, the ROC curves are shown in Figure 7. The areas under the curve forIGFBP5 / LTB4DH, MDK / LTB4DH and HoxAI3 / LTB4DH are 0.9223, 0.9199, and 0.7497, respectively. These areas, which measure the accuracy of the test, indicate that all three rates with LTB4DH are useful tests, in particularIGFBP5 / LTB4DH and MDK / LTB4DH.

To increase the sensitivity and specificity of CCT detection, combinations of two tests were used. Optional sensitivities and specificities of these test combinations are shown in Figure 6b. Figure 8a-f shows data separation in two-dimensional space for each of the three tests using LTB4DH and BAG1. These data show that the combination of two or more tests including either the down-regulated LTB4DH or BAG1 BTMs are capable of obtaining sensitivities and specificities greater than 90%. In addition, because these test the measurement of single gene expression signatures and not absolute levels of markers, they will be strong for variation in urine concentration.

To demonstrate the strength of tests involving ratios ratios to LTB4DH for urine concentration, IGFBP5 alone (ACt) and IGFBP5 / LTB4DH levels were plotted as a function of urine concentration (Figure 9a-b) and trend-adjusted data. It can be observed that for both urine samples from non-malignant controls and patients with TCC1 there is a decrease in IGFBP5 ACt with increasing urine concentration that is absent in the IGFBP5 / LTB4DH ratio. The effect is most evident with non-malignant samples because of the absence of other influences such as tumor size and heterogeneity in IGFBP5 and LTB4DH expression.

In some cases, when single markers are used in bladder cancer assays, the urine sample collection method may determine the amount of marker detected due to variations in the number of exfoliated bladder cells collected. This trend can lead to false positive or false negative results in a small proportion of samples. Using rates including LTB4DH or other low expression genes would provide a method to compensate for different methods. To test this hypothesis, samples collected from CBT patients by simple emptying (nine samples) or catheterization (28 samples) were tested for the presence of TCC and LTB4DH markers. Analysis of CBT markers only showed that deflated samples were more heavily represented at the lower end of the data range (upper Ct), according to the lower mean number of exfoliated cells in these samples compared to the catheterized samples. This is illustrated in the self-dispersing plots for IGFBP5, MDK and Ho-xA13 in Figure 10a-c. In contrast, when rates between these markers and LTB4DH were calculated, the emptied and catheterized samples were propagated over similar Ct bands (Figure 10d-f), illustrating that the calculation of gene expression signatures between the expression markers. High and low expression markers such as LTB4DH compensate for variations in marker levels introduced by different urine sampling methodologies.

Samples and urine from patients with low-grade tumors usually borderline in their BTM accumulation due to the presence of only small numbers of exfoliated cells in these samples. These samples are therefore at high risk of being misclassified due to variations in the sampling method or urine concentration.

The utility of gene expression rates incorporating down-regulated genes for CCT detection is therefore likely to be stated when applied for low grade CCT detection. To demonstrate this effect, a cohort of 43 low-grade urine specimens from patients with 123 low-grade CBT were tested with the IGFBP5, HoxAI 3, and LTB4DH markers. The clinical characteristics of the cohort are summarized in Fig. 13. The qPCR data for IGFBP5 and HoxAI3 were analyzed alone and at LTB4DH rates using the ROC curve area as a test precision measure (STATA statistical package). . The results are summarized in Fig. 14. Using the IGFBP5 marker, LTB4DH increased the detection accuracy of low grade (1-2 grade) TCCs by 9% and low grade TCCs of either internship at 8%. HoXAI 3 test accuracy of low grade Ta TCCs was increased by 3%.

Linear Discriminated Analysis of aPCR Data Using LTB4DH

Linear Discriminated Analysis (LDA) is a statistical technique (FI-sher RA "The Use of Multiple Measurements in Taxonomic Problems", Annals of Eugenics 7 179 (1936)) in which a linear combination of variables is generated such that there is Maximum separation between two or more groups. This linear combination of variables is called the "linear discriminant", which is a linear function of the input variables that maximally separates the classes from the data group. The ability of LDA (or any other classification technique) to characterize a particular data group, such as qPCR, can be tested using inter-validation. In this method, part of the data group is used to generate a classifier, and part of the data group is used to measure the effectiveness of that classifier. Data group division into training and test groups can be repeated multiple times (each time generating a new classifier). In k-fold inter-validation, the data group is divided from k to k, and each subgroup is used as the test group in one of k training and validation cycles. This can be extended to inter-validation excluding one (LOOCV) where each sample is classified according to a classifier generated from the remaining samples in the data group ("excluding one"; excluding a sample being tested).

LDA and LOOCV were used to illustrate the utility of TMsub-regulated LTB4DH in improving the diagnosis of CBT. qPCR was first performed on the control cohort and CBT urine specimens described in figure 13 that were supplemented with an additional 30 step 3 tumors (5> stage 1, 13 = stage 1,4 = Tis, and 8 = Ta ). Combinations of the LTB4DH, MDK, IGFBP5, H0XA13, T0P2a and CDC2 genes were tested for classifier performance, as evaluated by LOOCV. Later probability (that the "excluded" sample was a TCC sample) was used to generate ROC curves using the RO-CR package of the R statistical programming environment. The sensitivity of the classifier for a particular specificity was obtained. by reference to the appropriate DECR curve.

TCC detection sensitivity using combinations of over-regulated BTMs with and without LTB4DH was determined at a specificity of 85%. The results of this analysis are shown in figure 15. Addition of LTB4DH to assays including combinations of MDK, IGFBP5, Top2a, cdc2, and Ho-xA13 overregulated BTMs can be observed to increase overall sensitivity by 1-2% and detection sensitivity of Ta-stage, grade 1 tumors. 2 and grade 3 tumors by up to 3%.

Herein in the foregoing description reference has been made to integers or components having known equivalents, such equivalents are incorporated herein as if individually mentioned.

While the invention has been described by way of example and with reference to possible embodiments thereof, it should be appreciated that improvements and / or modifications may be made without departing from the scope or spirit of the invention.

INDUSTRIAL APPLICABILITY

Methods for detecting members of the TM family include nucleic acid, protein and peptide detection using demicroarrangement and / or real-time PCR methods. The compositions and methods of this invention are useful in diagnosing disease, assessing the efficacy of therapeutics, and for producing reagents and test kits suitable for measuring expression of TM family members.

Claims (27)

A method for determining the presence of a cancer in an individual, comprising: (a) providing a sample from the individual, (b) detecting the level of expression of at least two TM family members in said sample, wherein at least At least one TM is a sub-expressed TM, (c) establish whether the patient has cancer according to a predetermined threshold. The method of claim 1, wherein step (c) is performed by determining the expression rate of said BTMs. The method of claim 1, wherein step (c) is performed by performing regression analysis or classification on TM expression values. The method of claim 1, wherein the TM is a TM. The method of claim 1, wherein said cancer is bladder cancer. The method of claim 1, wherein at least one of the TMs is an overexpressed TM. A method according to claim 1, wherein at least one sub-expressed TM is a TM selected from the group outlined in Figure 3. The method of claim 1, wherein at least one subexpressed TM is a TM selected from the group outlined in Figure 4. The method according to claim 6, wherein the overexpressed TM is selected from the group outlined in Figure 11 or Figure 12. The method of claim 1, wherein said detecting step is performed by detecting TM mRNA expression. The method of claim 1, wherein said detecting step is performed by detecting expression of a deTM protein. The method of claim 1, wherein said detecting step is performed by detecting expression of a TM peptide. The method of claim 1, wherein the sample is any of biopsy, blood, serum, peritoneal washes, cerebrospinal fluid, urine and evacuation samples. A device for detecting a TM, comprising: a substrate having a TM capture reagent therein; and a detector associated with said substrate, said detector is capable of detecting a TM associated with said capture reagent, wherein the TM is an underexpressed TM. The device of claim 14, wherein the TM is a TM. The device of claim 14, wherein the TM capture ditoreagent is an oligonucleotide. The device of claim 14, wherein the TM capture ditoreagent is an antibody. The device of claim 14, wherein the TM is a TM selected from the group outlined in Figure 3. The device of claim 14, wherein the TM is a TM selected from the group outlined in Figure 4. A kit for determining the presence of a cancer in an individual, comprising: a substrate, at least two TM capture reagents, wherein at least one TM is an underexpressed TM; instructions for use. The kit of claim 20, wherein the TM is a TM. The kit of claim 20, wherein said TM capture reagent is a TM specific oligonucleotide. The kit of claim 20, wherein said TM capture reagent is a TM specific antibody. The kit of claim 20, wherein the subpressure TM is a TM selected from the group outlined in Figure 3. The kit of claim 20, wherein the subpressure TM is a TM selected from the group outlined in Figure 4. The kit of claim 20, wherein at least one TM is an overexpressed TM. The kit of claim 26, wherein the overpressure TM is a TM selected from the group outlined in Figure 11 or Figure 12.